As interests increase in oligonucleotide therapeutics, there has been a greater need for analytical techniques to properly analyze and quantitate these biomolecules. This article looks into some of the existing chromatographic approaches for oligonucleotide analysis, including anion exchange, hydrophilic interaction liquid chromatography, and ion pair chromatography. Some of the key advantages and challenges of these chromatographic techniques are discussed. Colloid formation in mobile phases of alkylamines and fluorinated alcohols, a recently discovered analytical challenge, is discussed. Mass spectrometry is the method of choice to directly obtain structural information about oligonucleotide therapeutics. Mass spectrometry sensitivity challenges are reviewed, including comparison to other oligonucleotide techniques, salt adduction, and the multiple charge state envelope. Ionization of oligonucleotides through the charge residue model, ion evaporation model, and chain ejection model are analyzed. Therapeutic oligonucleotides have to undergo approval from major regulatory agencies, and the impurities and degradation products must be well-characterized to be approved. Current accepted thresholds for oligonucleotide impurities are reported. Aspects of the impurities and degradation products from these types of molecules are discussed as well as optimal analytical strategies to determine oligonucleotide related substances. Finally, ideas are proposed on how the field of oligonucleotide therapeutics may improve to aid in future analysis.

Varicella zoster virus (VZV) establishes lifelong infection after primary disease and can reactivate. Several drugs are approved to treat VZV diseases, but new antivirals with greater potency are needed. Previously, we identified), which had significant anti-VZV activity. In this communication, we report the synthesis and evaluation of numerous L-BHDU prodrugs: amino acid esters (14−26), phosphoramidates (33−34), long-chain lipids (ODE-L-BHDU-MP, 38, and HDP-L-BHDU-MP, 39), and phosphate ester prodrugs (POM-L-BHDU-MP, 41, and POC-L-BHDU-MP, 47). The amino acid ester L-BHDU prodrugs (L-phenylalanine, 16, and L-valine, 17) had a potent antiviral activity with EC 50 values of 0.028 and 0.030 μM, respectively. The phosphate ester prodrugs POM-L-BHDU-MP and POC-L-BHDU-MP had a significant anti-VZV activity with EC 50 values of 0.035 and 0.034 μM, respectively, and no cellular toxicity (CC 50 > 100 μM) was detected. Out of these prodrugs, ODE-L-BHDU-MP (38) and POM-L-BHDU-MP (41) were selected for further evaluation in future studies.